Mutations in PYCR2, encoding pyrroline-5-carboxylate reductase 2, cause microcephaly and hypomyelination

Abstract

Despite recent advances in understanding the genetic bases of microcephaly, a large number of cases of microcephaly remain unexplained, suggesting that many microcephaly syndromes and associated genes are yet to be identified. Here we report mutations in PYCR2, which encodes an enzyme in the proline biosynthesis pathway, as the cause of a unique syndrome characterized by postnatal microcephaly, hypomyelination, and reduced cerebral white matter volume. Linkage mapping and whole-exome sequencing identified homozygous mutations in PYCR2 (c.355C>T [p.Arg119Cys] and c.751C>T [p.Arg251Cys]) in the affected individuals of two consanguineous families. A lymphoblastoid cell line from one affected individual showed a strong reduction in PYCR2 level. When mutant cDNAs were transfected into HEK293FT cells, the mutant protein retained normal mitochondrial localization but the level was lower than the wild-type protein, suggesting that mutant protein is less stable. A PYCR2-deficient HEK293FT cell line generated by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing showed that PYCR2 loss of function led to decreased mitochondrial membrane potential and increased susceptibility to apoptosis under oxidative stress. Morpholino-based knockdown of the zebrafish PYCR2 homolog, pycr1b, recapitulated the human microcephaly phenotype, which was rescued by wild-type human PYCR2 mRNA, but not by mutant mRNAs, further supporting the pathogenicity of the identified variants. Hypomyelination and the absence of lax, wrinkly skin distinguishes this condition from that caused by previously reported mutations in the gene encoding PYCR2’s isozyme, PYCR1, suggesting a unique and indispensable role for PYCR2 in the human central nervous system during development.